Controlling stem cell divisions in time and space

Animals and plants are multicellular (metazoan), and are made up of cells descended from a single original cell, the fertilized egg. Complex organisms such as humans are composed of trillions of cells, whereas simple organisms may consist of just a few hundred cells. During development from egg to adult, cells of all metazoan organisms therefore need to divide, or proliferate, in order to increase in number. However, it is equally as important that cells stop proliferating at the appropriate time and differentiate (take on specialised roles appropriate to their individual circumstances of position, ancestory and timing). Proliferation and differentiation therefore need to be in perfect balance to ensure that tissues and organs develop with the correct size and functionality. Stem cells have particularly important properties. They have the ability to self-renew (make more of themselves) as well as give rise to daughters that differentiate. They do this by dividing asymmetrically: one daughter cell retains the stem cell fate of further proliferation, whereas the other daughter stops dividing in order to differentiate. Stem cells can also sometimes divide symmetrically, with both daughters retaining the ability to proliferate further, helping to expand the number of progenitor cells for particular purposes. Stem cells sometimes retain the ability to proliferate throughout the lifetime of the organism in order to generate (and sometimes re-generate) particular tissues and organs. For example, hematopoietic stem cells generate blood cells throughout the lifetime of many organisms, and epidermal stem cells constantly generate new skin cells. Stem cell divisions must therefore be very tightly controlled, so that the conflicting demands of proliferation vs. differentiation are correctly balanced. Understanding how stem cell divisions are controlled is crucial to 21st century biological research because it may be possible to harness these cells in order to produce any required cell type on demand, for example new spinal cord neurons for patients with spinal injury. It is also thought that stem cells are at the heart of carcinogenesis. However, we have much to understand about the basic biology of stem cells before potential therapies can become routine. All properties of cells are ultimately under genetic control, which dictates that the appropriate RNA and protein molecules are produced at the correct time and place. Human development is an incredibly complicated process, involving trillions of cells and many thousands of genes, therefore before we can begin to understand human development and human stem cells, we need to investigate a simpler system. This is where the tiny worm Caenorhabditis elegans comes in. Many of the genes that are important for C. elegans development are remarkably similar to human genes, therefore studying C. elegans development will ultimately shed light on the biology of higher animals including humans. C. elegans have cells called seam cells. These are stem cells, in that they divide throughout development to give rise to more stem cells, as well as daughters that differentiate by becoming skin cells and neurons. Our aim is to understand which genes are important for controlling the correct pattern of seam cell divisions. Previously, we have isolated two genes, rnt-1 and bro-1, as being important for this process. Strikingly, the human counterparts of both these genes, Runx and CBFb, are also involved in stem cell development, including haematopoietic and epidermal stem cells. This means we are studying a conserved process, and lessons gained from studying the problem in C. elegans are very likely to be applicable to higher organisms including humans. In this proposal, we are seeking to understand more about how rnt-1 and bro-1 control seam cell divisions, and also to isolate new genes involved in this process.

This application proposes to use C. elegans as model system for studying the control of stem cell division patterns. During development, cells must stop proliferating and differentiate at the appropriate time. This decision is particularly pertinent in stem cells, which both self-renew and give rise to differentiated cells that can generate particular cell types over the lifetime of an organism. In C. elegans, the seam cells offer a paradigm for the stem cell mode of division. They are multipotent neuroectodermal cells that divide symmetrically to expand the pool of progenitor cells, and asymmetrically to contribute various differentiated cell types (as well as more stem cells). Our previous work has sought to analyse mutants with defects in seam cell proliferation and differentiation. We have identified rnt-1, a homologue of the Runx family of transcription factors, and bro-1, a CBFb (Runx DNA binding partner) homologue, as being key players in the decision between proliferation and differentiation. Loss of function of bro-1 or rnt-1 results in a failure of seam cell proliferation whereas over-expression results in too much proliferation, at the expense of differentiation. The experiments described in this proposal build on a substantial body of pilot data aimed at identifying the molecular mechanisms responsible for the correct dynamic regulation of rnt-1 and bro-1 during development. Methods proposed include mutant/RNAi analysis of gene function, lineage analysis, use of transgenic gene expression reporter strains/ immunofluorescence, and site directed mutagenesis of regulatory regions. We also aim to use targeted RNAi screens to identify novel pathways operating in parallel to rnt-1/bro-1 to regulate stem cell division patterns. Given that Runx/CBFb genes are also heavily involved in regulating stem cell lineages in mammalian systems, for example in haematopoietic stem cells, we expect knowledge gained from C. elegans to be generally applicable.